1. Field of the Invention
The present invention relates to an in-vivo examination method and an in-vivo examination apparatus that allow in-vivo examination of tissue of a living organism.
The present invention also relates to an objective lens, a microscope apparatus, and a microscopy method.
This application is based on Japanese Patent Applications, Nos. 2005-369763 and 2006-046625, the contents of which are incorporated herein by reference.
2. Description of Related Art
Conventionally, in-vivo examination of tissue of a living organism has been carried out by making a large incision in the organism to expose the site to be examined or by excising tissue including the site to be examined.
In particular, for brain examination, an apparatus and method for examining brain tissue by thinning the cranial bone and employing multiphoton excitation {are used (for example, refer to Japanese Translation of PCT International Application, Publication No. 2004-518488).
A technology for carrying out fluoroscopy on the brain of a living organism by using an optical fiber, instead of an objective optical system, is also known (for example, refer to Japanese Unexamined Patent Application, Publication No. 2000-262460).
When carrying out such examinations, for example, mouse-brain researchers specializing in central nervous system pharmacology or neurophysiology carry out experiments by locating regions in the actual brain tissue on the basis of data about the tissue and anatomy of small laboratory animals, such as mice, arranged into an atlas.
However, when a large incision is made in an organism for examination, there is a problem in that the condition of an organism in which an incision is made differs from the condition of an organism in which no incision is made. Furthermore, when tissue is excised for examination, there is a problem is that the organism cannot be examined in vivo.
There is also a problem in the examination method disclosed in Japanese Translation of PCT International Application, Publication No. 2004-518488 in that tissue in deep regions of the brain cannot be examined.
According to the examination method disclosed in Japanese Unexamined Patent Application, Publication No. 2000-262460, an examination site is determined only on the basis of a detected fluorescence image of a specific fluorochrome-labeled site. Thus, the general location of the examination site can be detected, but it is difficult to determine whether or not the detected area is the actual site desired to be examined. Furthermore, depending on the position and direction of insertion of the optical fiber, there is a possibility that the site to be examined cannot be reached. In some cases, the optical fiber may pass through the examination site and damage the tissue at the examination site.
A microscope system suitable for in-vivo examination of animals is known (for example, refer to Japanese Unexamined Patent Application, Publication No. 2005-241671).
This microscope system includes an objective lens having a small-diameter end section that can be inserted into the body of an animal in a less invasive manner. The microscope system employs a confocal microscopy method in which the end surface of the objective lens is positioned close to or pressed against the examination site in the body of the animal and excitation light, such as laser light, is emitted, and the fluorescence that returns from the examination site is observed.
In such a microscope system, the examination site has be to positioned extremely close to the end surface of the objective lens because the working distance of an objective lens having a small-diameter end section is extremely small, i.e., several hundred micrometers, and the depth of field is extremely shallow because it employs a confocal microscope. Therefore, when the examination site can be positioned sufficiently close to the end surface of the objective, a clear fluorescence image can be acquired.
However, when such a microscope system is used for examination of a lumen, the following problems occur.
In other words, when the small-diameter end section of the objective lens is inserted into a lumen of a laboratory animal, the tip of the small-diameter end section is positioned in the hollow space of the lumen and is not positioned close to the inner wall of the lumen. Therefore, it is difficult to focus at the examination site, such as a tumor formed on the inner wall of the lumen.
In such a case, if the examination site is relatively soft, the lumen in the laboratory animal's body can be deformed by applying external pressure to the animal's body so as to move the inner wall of the lumen closer to the tip of the small-diameter end section. However, when the lumen is positioned deep inside the body, it is difficult to apply pressure to the lumen. In addition, even when the examination site is temporarily or unintentionally focused, it is difficult to stably maintain the focused state for a long period of time.